Jennifer A. Mitchell

The Air Noncoding RNA Epigenetically Silences Transcription by Targeting G9a to Chromatin

A number of large noncoding RNAs (ncRNAs) epigenetically silence genes through unknown mechanisms. The Air ncRNA is imprinted—monoallelically expressed from the paternal allele. Air is required for allele-specific silencing of the cis-linked Slc22a3, Slc22a2, and Igf2r genes in mouse placenta. We show that Air interacts with the Slc22a3 promoter chromatin and the H3K9 histone methyltransferase G9a in placenta. Air accumulates at the Slc22a3 promoter in correlation with localized H3K9 methylation and transcriptional repression. Genetic ablation of G9a results in nonimprinted, biallelic transcription of Slc22a3. Truncated Air fails to accumulate at the Slc22a3 promoter, which results in reduced G9a recruitment and biallelic transcription. Our results suggest that Air, and potentially other large ncRNAs, target repressive histone-modifying activities through molecular interaction with specific chromatin domains to epigenetically silence transcription.

Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells

The discovery of interchromosomal interactions in higher eukaryotes points to a functional interplay between genome architecture and gene expression, challenging the view of transcription as a one-dimensional process. However, the extent of interchromosomal interactions and the underlying mechanisms are unknown. Here we present the first genome-wide analysis of transcriptional interactions using the mouse globin genes in erythroid tissues. Our results show that the active globin genes associate with hundreds of other transcribed genes, revealing extensive and preferential intra- and interchromosomal transcription interactomes. We show that the transcription factor Klf1 mediates preferential co-associations of Klf1-regulated genes at a limited number of specialized transcription factories. Our results establish a new gene expression paradigm, implying that active co-regulated genes and their regulatory factors cooperate to create specialized nuclear hot spots optimized for efficient and coordinated transcriptional control.

REPLICATION AND TRANSCRIPTION: SHAPING THE LANDSCAPE OF THE GENOME

As the relationship between nuclear structure and function begins to unfold, a picture is emerging of a dynamic landscape that is centred on the two main processes that execute the regulated use and propagation of the genome. Rather than being subservient enzymatic activities, the replication and transcriptional machineries provide potent forces that organize the genome in three-dimensional nuclear space. Their activities provide opportunities for epigenetic changes that are required for differentiation and development. In addition, they impose physical constraints on the genome that might help to shape its evolution.

Transcription factories are nuclear subcompartments that remain in the absence of transcription.

Nascent transcription occurs at nuclear foci of concentrated, hyperphosphorylated RNA polymerase II (RNAPII). We investigate RNAPII localization, distal gene co-association, and Hbb locus conformation during inhibition of transcription. Our results show distal active genes remain associated with RNAPII foci and each other in the absence of elongation. When initiation is inhibited, active genes dissociate from RNAPII foci and each other, suggesting initiation is necessary to tether distal active genes to shared foci. In the absence of transcription RNAPII foci remain, indicating they are not simple accumulations of RNAPII on transcribed genes but exist as independent nuclear subcompartments.

The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements

The mammalian genome harbors up to one million regulatory elements often located at great distances from their target genes. Long-range elements control genes through physical contact with promoters and can be recognized by the presence of specific histone modifications and transcription factor binding. Linking regulatory elements to specific promoters genome-wide is currently impeded by the limited resolution of high-throughput chromatin interaction assays. Here we apply a sequence capture approach to enrich Hi-C libraries for >22,000 annotated mouse promoters to identify statistically significant, long-range interactions at restriction fragment resolution, assigning long-range interacting elements to their target genes genome-wide in embryonic stem cells and fetal liver cells. The distal sites contacting active genes are enriched in active histone modifications and transcription factor occupancy, whereas inactive genes contact distal sites with repressive histone marks, demonstrating the regulatory potential of the distal elements identified. Furthermore, we find that coregulated genes cluster nonrandomly in spatial interaction networks correlated with their biological function and expression level. Interestingly, we find the strongest gene clustering in ES cells between transcription factor genes that control key developmental processes in embryogenesis. The results provide the first genome-wide catalog linking gene promoters to their long-range interacting elements and highlight the complex spatial regulatory circuitry controlling mammalian gene expression.

Progesterone and Gravidity Differentially Regulate Expression of Extracellular Matrix Components in the Pregnant Rat Myometrium

Abstract Myometrial growth and remodeling during pregnancy depends on increased synthesis of interstitial matrix proteins. We hypothesize that the presence of mechanical tension in a specific hormonal environment regulates the expression of extracellular matrix (ECM) components in the uterus. Myometrial tissue was collected from pregnant rats on Gestational Days 0, 12, 15, 17, 19, 21, 22, 23 (labor), and 1 day postpartum and ECM expression was analyzed by Northern blotting. Expression of fibronectin, laminin β2, and collagen IV mRNA was low during early gestation but increased dramatically on Day 23 during labor. Expression of fibrillar collagens (type I and III) peaked Day 19 and decreased near term. In contrast, elastin mRNA remained elevated from midgestation onward. Injection of progesterone (P4) on Days 20–23 (to maintain elevated plasma P4 levels) delayed the onset of labor, caused dramatic reductions in the levels of fibronectin and laminin mRNA, and prevented the fall of collagen III mRNA levels on Day 23. Treatment of pregnant rats with the progesterone receptor antagonist RU486 on Day 19 induced preterm labor on Day 20 and a premature increase in mRNA levels of collagen IV, fibronectin, and laminin. Analysis of the uterine tissue from unilaterally pregnant rats revealed that most of the changes in ECM gene expression occurred specifically in the gravid horn. Our results show a decrease in expression of fibrillar collagens and a coordinated temporal increase in expression of components of the basement membrane near term associated with decreased P4 and increased mechanical tension. These ECM changes contribute to myometrial growth and remodeling during late pregnancy and the preparation for the synchronized contractions of labor.

Differential expression of activator protein-1 transcription factors in pregnant rat myometrium.

Abstract While the AP-1 (activator protein-1) genes c-fos and c-jun have been implicated in the expression of myometrial genes associated with the onset of labor, there are no data concerning the role of other members of this family of transcription factors. To address this issue, we defined the expression and hormonal regulation of AP-1 genes in the rat myometrium during pregnancy and labor. Tissue was collected on Days 12, 15, 17, 19, 21, 22, and 23 (labor) and 1 day postpartum. Expression of c-fos, fosB, fra-1, fra-2, and junB was low during early gestation, with a 5- to 10-fold increase on Day 23 during labor, and returned to low levels 1 day postpartum. In contrast, the levels of c-jun and junD remained relatively constant throughout gestation. Administration of progesterone (P4; 16 mg/kg s.c./day) beginning on Day 20 (to maintain elevated plasma P4 levels) prevented the onset of labor and blocked the expected rise in c-fos, fosB, fra-1, fra-2, and junB expression on Day 23. In contrast, administration of the progesterone receptor antagonist RU486 (10 mg/kg s.c.) on Day 19 induced preterm labor and a premature increase in mRNA levels of c-fos, fra-1, fra-2, and junB. In unilaterally pregnant rats, stretch imposed by the growing fetus was found to increase the expression of c-fos, fosB, fra-1, fra-2, and junB only in the gravid horn on the day of labor. These data raise the possibility that AP-1 transcription factors integrate endocrine and mechanical signals, leading to myometrial gene expression required for uterine remodeling and the initiation of labor.

A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential

The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Two gene-proximal enhancers, Sox2 regulatory region 1 (SRR1) and SRR2, display activity in reporter assays, but deleting SRR1 has no effect on pluripotency. We identified and functionally validated the sequences required for Sox2 transcription based on a computational model that predicted transcriptional enhancer elements within 130 kb of Sox2. Our reporter assays revealed three novel enhancers--SRR18, SRR107, and SRR111--that, through the formation of chromatin loops, form a chromatin complex with the Sox2 promoter in ES cells. Using the CRISPR/Cas9 system and F1 ES cells (Mus musculus(129) × Mus castaneus), we generated heterozygous deletions of each enhancer region, revealing that only the distal cluster containing SRR107 and SRR111, located >100 kb downstream from Sox2, is required for cis-regulation of Sox2 in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression, and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together, these data identify a distal control region essential for Sox2 transcription in ES cells.

Enhancer identification in mouse embryonic stem cells using integrative modeling of chromatin and genomic features

BackgroundEpigenetic modifications, transcription factor (TF) availability and differences in chromatin folding influence how the genome is interpreted by the transcriptional machinery responsible for gene expression. Enhancers buried in non-coding regions are found to be associated with significant differences in histone marks between different cell types. In contrast, gene promoters show more uniform modifications across cell types. Here we used histone modification and chromatin-associated protein ChIP-Seq data sets in mouse embryonic stem (ES) cells as well as genomic features to identify functional enhancer regions. Using co-bound sites of OCT4, SOX2 and NANOG (co-OSN, validated enhancers) and co-bound sites of MYC and MYCN (limited enhancer activity) as enhancer positive and negative training sets, we performed multinomial logistic regression with LASSO regularization to identify key features.ResultsCross validations reveal that a combination of p300, H3K4me1, MED12 and NIPBL features to be top signatures of co-OSN regions. Using a model from 10 signatures, 83% of top 1277 putative 1 kb enhancer regions (probability greater than or equal to 0.8) overlapped with at least one TF peak from 7 mouse ES cell ChIP-Seq data sets. These putative enhancers are associated with increased gene expression of neighbouring genes and significantly enriched in multiple TF bound loci in agreement with combinatorial models of TF binding. Furthermore, we identified several motifs of known TFs significantly enriched in putative enhancer regions compared to random promoter regions and background. Comparison with an active H3K27ac mark in various cell types confirmed cell type-specificity of these enhancers.ConclusionsThe top enhancer signatures we identified (p300, H3K4me1, MED12 and NIPBL) will allow for the identification of cell type-specific enhancer regions in diverse cell types.

Regulation of connexin43 expression by c-Fos and c-Jun in myometrial cells

Labor is associated with a dramatic increase in the myometrial expression of connexin43 (Cx43) which is thought to mediate myocyte contractile coupling. The transcription factor c-fos is also dramatically increased prior to the onset of labor and is therefore a potential regulator of Cx43. The promoter region of Cx43 contains a conserved activator protein-1 (AP-1) site, which binds dimers of Fos and Jun proteins. We constructed expression vectors for c-Fos and c-Jun to investigate the role of these transcription factors in the regulation of Cx43 expression. These expression vectors were then co-transfected into SHM (syrian hamster myocyte) cells with a Cx43 promoter-Luciferase vector. The combinations of c-Fos and c-Jun proteins activated the Cx43 promoter while c-Jun alone had no effect on Cx43 promoter activity. Mutation of the AP-1 site was found to reduce this responsiveness. These data indicate that the transcription factors c-Fos and c-Jun are important in the regulation of Cx43 expression.